Amphiregulin (AR) is a member of the epidermal growth factor (EGF)/transforming growth factor alpha (TGFalpha) gene family since it structurally resembles EGF or TGFalpha, and it can bind to the EGF receptor. MCF10A cells are a spontaneously immortalized, nontransformed human mammary epithelial cell line that requires EGF for growth. AR (0.05-0.5nM) was found to stimulate the growth of MCF10A cells to a level that was comparable to the growth stimulation produced by equivalent concentrations of EGF. MCF10A cells express low levels of a 1.4kb mRNA transcript for AR. MCF10A cells transformed by either an activated human c-Ha-ras protooncogene or with a nonactivated overexpressed rat c-neu (erb B-2) gene exhibited a 15-30 fold increase in the level of AR mRNA expression. Endogenous AR protein could be immunocytochemically detected in MCF10A cells using a polyclonal antibody obtained after immunizing rabbits with a 19-mer peptide corresponding to the NH2 terminus of AR. MCF10A parental cells exhibited a weak cytoplasmic staining with this antibody whereas c-Ha- ras or c-erb B-2 transformed MCF10A cells differ from the pattern of endogenous TGFalpha expression which was enhanced in c-Ha-ras but not in c-erb B-2 transformed MCF10A cells. In order to ascertain what role AR might perform in MCF10A transformed cells, we synthesized two different phosphorothioate 20-mer antisense oligodeoxynucleotides complementary to the 5' sequence of AR mRNA starting respectively 3 and 9 nucleotides upstream of the AUG translation initiation codon. These antisense oligodeoxynucleotides and a 20-mer missense oligonucleotide at a concentration of 10 uM were able to inhibit the soft agar growth of c- Ha-ras and c-erb B-2 transformed cells by 70-80% as compared to the control missense treated cells. These data suggest that AR may function as an autocrine growth factor in mammary epithelial cells transformed by ras and erb B-2 oncogenes and that these oncogenes can differentially modulate the expression of various members of the EGF family.